Summary of Work: Estrogen receptors are members of the ligand-dependent transcription factor super-family. Male mice homozygous for a mutation in the estrogen receptor-alpha gene (alpha-ERKO mice) are infertile. However, male mice homozygous for a mutation in the estrogen receptor-beta gene (beta-ERKO) are fertile, indicating that estrogen acts on the male reproductive system primarily through estrogen receptor-alpha. The goals of this project were to determine the role of estrogen in male germ cell development, the causes of infertility in male alpha-ERKO mice, and the identity of estrogen-regulated genes in the testis and epididymis. It was unknown if male alpha-ERKO mice are infertile due to disruption of estrogen-regulated processes within spermatogenic cells, associated somatic cells, or both. We hypothesized that estrogen receptors do not directly regulate genes within developing germ cells, but are required by somatic cells in the testis and/or epididymis. We addressed this by transplanting spermatogenic cells from the testes of alpha-ERKO+/- or alpha-ERKO-/- donor mice to those of isogenic wild-type mice that had been treated with busulphan to eliminate most endogenous spermatogenic cells. After allowing regeneration of spermatogenesis, recipients were mated with wild-type females. The alpha-ERKO donor mice were C57Bl/6J (black), the male recipients were albino C57BL/6J, and the wild-type females were albino CD-1. This allowed the use of coat color markers to identify offspring produced from transplanted germ cells (black) and from regenerating endogenous spermatogenic cells (white). Litters of mice were sired by recipients that received germ cells from alpha-ERKO-/- donors, including some with all white pups, some with black pups and white pups, and some with only black pups. These results support the hypothesis that male germ cells do not directly require estrogen receptor-alpha for development, and suggest that alpha-ERKO males are infertile because of functional deficiencies in somatic cells of the testis and/or epididymis. Estrogen receptor-alpha is present in the somatic cells of the male reproductive tract, with the highest levels of expression occurring in the epithelial cells of the efferent ducts and the caput epididymis. The mammalian epididymis functions as a storage site and is responsible for the final maturation of spermatozoa. During the approximately two-week period of transit through the epididymis, spermatozoa undergo physiological changes whereby they acquire the ability to perform effective forward motility and become competent to fertilize an oocyte. Epididymal sperm of alpha-ERKO mice fail to undergo these maturational changes. This suggests that estrogen receptor-alpha plays a fundamental role in male reproduction by regulating critical aspects of epididymal sperm maturation. We hypothesized that estrogen regulates the synthesis of proteins by epididymal cells required for sperm to undergo the functional changes and surfaces modifications necessary to become capable of fertilization. We used mouse microarrays produced in the NMC to identify genes expressed at significantly different levels in the epididymis of alpha-ERKO mice and wild-type mice as an approach to identifying functional changes that cause infertility in alpha-ERKO males. Several genes were identified that are predicted to be involved in sperm maturation and are being analyzed. The microarray results for most of these genes were confirmed by northern analysis and real-time PCR. However, the changes in the level of gene expression were modest. Additional real-time PCR studies are underway to determine if expression of these genes varies among the different regions of the epididymis or at different ages in male alpha-ERKO and wild-type mice. Significance: There is considerable controversy over the possible role of environmental estrogens or endocrine disruptors in the reported decline by one half in semen volume and sperm numbers, and the doubling of occurrence of testicular cancers and reproductive tract malformations in human males over the last five decades. The debates surrounding these issues have made it obvious that relatively little is known about the role of estrogen in male reproduction. Our studies were the first to show definitively that estrogen is required for male reproductive function and indicate the importance of identifying the estrogen-dependent processes required for male fertility.